Method for the production of α(1→2) oligodextrans using Leuconostoc mesenteroides B-1299

ABSTRACT

A method for the production of oligodextrans containing an α(1→2) bond which uses glycosyltransferase from Leuconostoc mesenteroides, particularly strain B-1299, sucrose and an accepter moiety such as glucose, maltose, isomaltose, isomaltotriose or methyl α-glucose. Optionally, this product may be further treated with an endodextranase or glucosamylase.

The invention relates to a process for the enzymatic preparation ofoligodextrans used in the production of sugar substitutes and to noveloligodextrans.

It is known to prepare dextrans of high molar mass, greater than onemillion (degree of polymerization greater than 6000 units of glucose) bythe action of the lactic bacterium Leuconostoc mesenteroides on sucrose.It is also known that certain strains of this bacterium produce dextranscontaining glucoside α(1→2) bonds, which constitute the branching pointsin these dextrans.

It was not known, however, prior to the present invention to carry outthe enzymatic synthesis of oligodextrans (dextrans with a low degree ofpolymerization) containing at least one glucoside α(1→2) bond directlyusing sucrose and a sugar acceptor of glucose residues originating fromsucrose.

The object of the present invention is to provide just such a process.

More precisely, the invention relates to a process for the preparationof a mixture of oligodextrans containing at least one α(1→2) glucosidebond and containing a major proportion of oligodextrans of the generalformula (O-α-D-glucopyranosyl-(1→2))_(m)(O-α-D-glucopyranosyl-(1→6))_(n) A where A is the residue of a sugaracceptor of glucose chosen from amongst maltose, isomaltose,isomaltotriose, methyl α-glucoside and glucose, m is from 1 to 10 and nis from 1 to 30, the position of the α(1→2) glucoside bond or bondsbeing arbitrary, characterized in that sucrose and a sugar acceptor ofglucose chosen from the group comprising maltose, isomaltose,isomaltotriose, methyl α-glucoside and glucose are brought into contactin the presence of glucosyltransferase enzyme extracted from at leastone strain of the lactic bacterium Leuconostoc mesenteroides capable ofproducing, by fermentation, a dextran containing α(1→2) glucoside bonds,for approximately 2 to 48 hours in an aqueous medium.

The position of the glucoside α(1→2) bond or bonds in the formulaindicated above is variable as a function, in particular, of the natureof group A and of the molar mass of the oligodextran, which itself isdependent on the reaction conditions. These bonds may, for example, beon the main chain, on the branches or may constitute the branchingpoints in the oligodextran.

Non-restrictive examples of suitable strains of the lactic bacteriumLeuconostoc mesenteroides are the following: NRRL B-1299, B-1399,B-1397, B-1298, B-1396, B-1424, B-1382, B-1149 and B-523.

Some of the oligodextrans (sometimes also called oligosaccharides)produced by the process of the invention are novel compounds. Thepreparation ofO-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl-α(1→6)-D-glucosetrisaccharide by acetolysis of dextran NRRL B-1397 has, however, beendescribed by K. Sakakibara et al. in Carbohydrate Research, 25 (1972),pages 443-451. Likewise, Y. Mitsuishi et al., in Carbohydrate Research,127 (1984), pages 331-337, have described oligosaccharides branched bymeans of an α(1→2) glucoside bond.

The invention thus also relates to oligodextrans of the general formula(O-α-D-glucopyranosyl-(1→2))_(m) (O-α-D-glucopyranosyl-(1→6))_(n) Awhere A is the residue of a sugar acceptor of glucose chosen fromamongst maltose and methyl α-glucoside, m is from 1 to 3 and n is from 1to 10, the position of the α(1→2) glucoside bonds being arbitrary, asnovel products.

The oligodextrans produced by the process of the invention which containan α(1→2) glucoside bond which is located at their non-reducing end orwhich constitutes a branching point in the oligodextran, whether novelin themselves or not, are particularly resistant to enzymatic hydrolysisby glucohydrolase enzymes, such as endodextranase and glucoamylase, thisresistance being due to the presence of this rare α(1→2) glucoside bondlocated at their non-reducing end or constituting a branching point inthe oligodextran.

This property makes them useful as fillers or extenders in sugarsubstitutes which are metabolizable by man only slightly or not at all.They may therefore be used in low-calorie foodstuff formulations, mixedwith a strong sweetener, such as aspartame or equivalent.

The oligosaccharides of the invention may also promote the growth andthe development of certain beneficial microorganisms of the intestinalflora. This property may make them equally useful as additives in animalfeeds (additives of zootechnic value) and for human foodstuffs(dietetics and nutrition).

The invention thus also relates to the use of a mixture of oligodextranscontaining at least one α(1→2) glucoside bond located at theirnon-reducing end or constituting a branching point in the oligodextran,which contain a major proportion of oligodextrans of the general formula

    O-α-D-glucopyranosyl-(1→2).sub.m (O-α-D-glucopyranosyl-

    (1→6)).sub.n A

where A is the residue of a sugar acceptor of glucose chosen fromamongst maltose, isomaltose, isomaltotriose, methyl α-glucoside andglucose, m is from 1 to 10 and n is from 1 to 30, the position of theα(1→2) glucoside bond or bonds being arbitrary, as fillers in sugarsubstitutes or as foodstuff additives.

As indicated above, the enzymatic synthesis reaction is carried out inthe presence of glucosyltransferase enzyme (E.C:2.4.1.5) extracted fromstrains of the bacterium Leuconostoc mesenteroides capable of producing,by fermentation, a dextran containing α(1→2) glucoside bonds. Thisreaction may be represented by the global equation:

    glucosyltransferase

    sucrose+sugar acceptor→oligodextrans+fructose

Suitable strains are, inter alia, the NRRL B-1299, B-1399, B-1397,B-1298, B-1396, B-1424, B-1382, B-1149 and B-523 strains. These strainsare obtainable from the N.R.R.C. (Northern Regional Research Center),the address of which is as follows: Agricultural Research Service, U.S.Department of Agriculture, 1815 North University Street, Peoria, Ill.61604, USA. They were characterized at the start of the 1950's using aselection of microorganisms isolated from soil. They have been indexed,amongst others, in the following publication:

"Characterization and classification of dextrans from ninety-six strainsof bacteria",

A. Jeanes et al. (1954), J. Amer. Chem. Soc. 76, 5041-5052.

It goes without saying that it would also be possible to use, in placeof the strains cited above, strains derived from these and obtained bymutagenesis in a conventional manne, i.e. by irradiation of the strainsor by the action of a chemical agent on the said strains, followed byculture of the surviving individuals, or obtained by selection ofnatural mutants. For the purpose of the invention, these mutant strainsare considered as equivalent to the strains cited above.

Glucosyltransferase can be obtained by culturing an appropriate strainof the lactic bacterium Leuconostoc mesenteroides, such as the NRRLB-1299 strain, on a suitable nutrient medium containing, in particular,sucrose in order to induce the production of the glucosyltransferase.After growth of the bacterium, the enzymatic activity is extracted bythe addition of polyethylene glycol so as to precipitate and concentratethe various forms of glucosyltransferase: extracellular, bound to cellsand to insoluble polysaccharides, and intracellular. This enzymaticpreparation then contains whole cells of L. mesenteroides B-1299. Knowntechniques of cell breaking may be used at the end of the culture toaugment the enzymatic activity on the one hand, and to destroy the cellson the other hand (mechanical crushing, use of chemical agents orenzymatic agents (lysozyme, . . . )). However, this operation is notstrictly necessary. The extraction of the enzymatic activity bypolyethylene glycol is carried out a second time after havingredissolved the first precipitate (obtained after the first extraction),for example with the aid of a 20 mM sodium acetate buffer of pH 5.4,containing 0.02 g/l calcium chloride. The second precipitate containsall the enzymatic activity. It can be lyophilized or frozen inconcentrated form without loss of activity. Other purificationtechniques may be used, such as centrifugation, ultrafiltration or achromatographic process.

The enzymatic synthesis of oligodextrans is carried out with the aid ofthis enzymatic preparation or of a fraction of this preparationcorresponding to a specific form of the enzyme (intracellular enzyme,after breaking of the cells, extracellular enzyme bound to insolublepolymers, soluble extracellular enzyme) in the presence of sucrose,which constitutes the substrate of the reaction, and of a sugaracting--as a glucose acceptor, such as maltose (or a material rich inmaltose, such as a starch hyrolysis product), isomaltose, methylα-glucoside, isomaltotriose or glucose (or a material rich in glucose,such as a starch hydrolysis product).

By way of example, the reaction can be carried out at between 5° and 45°C., preferably between about 20° and 30° C. The pH of the reaction isbetween 4.5 and 7, preferably between 5 and 6. The reaction time isusually between about 2 and 48 hours; it depends on the concentration ofthe enzyme in the synthesis medium. Preferably, the enzyme concentrationis 0.20 to 1 U/ml, but it could be higher if desired. One enzymatic unit(U) is defined as the quantity of enzyme necessary to produce 1micromole of fructose per minute under the following standard conditionsfor the measurement of the activity:

sucrose: 100 g/l

20 mM sodium acetate buffer, pH 5.2

30° C.

The proportions of sucrose and of sugar glucose acceptor are not verycritical. By way of example, a sucrose concentration of from 20 to 600g/l and a sugar glucose acceptor concentration of from 10 to 300 g/l maybe used. The highest yields are obtained from the oligodextranssynthesis when the ratio of the sucrose concentration, expressed in g/l,to the acceptor concentration, expressed in g/l, is between 0.5 and 10and preferably between 2 and 4.

The enzyme may be used either in the free form (discontinuous process)or incorporated inside a cross-linked gel (calcium alginate gel, forexample) or bound in a covalent manner to an insoluble support. If theenzyme is immobilized, it may then be used in an appropriate reactor(fixed bed reactor, fluidized bed reactor, etc. . . . ) for thecontinuous production of oligodextrans.

After the action of the enzyme, the reaction medium may be analysed by ahigh performance liquid chromatography (HPLC) method with inverse phaseseparation of the oligodextrans (for example the micro-Bondapack C18column from Millipore-Waters), elution being effected with ultrapurewater or with a water/methanol mixture (% methanol between 0 and 6%(v/v)). This quantitative method enables all of the oligodextrans with adegree of polymerization of between about 2 and 20 to be separated.

The assay of the reducing sugars produced in the course of the reactionmay be effected with D.N.S. reagent (sodium dinitrosalicylate in analkaline medium).

The reaction mixture obtained comprises oligodextrans containing atleast one α(1→2) glucoside bond, oligodextrans which do not contain sucha bond, fructose and unaltered sugar glucose acceptor.

The oligodextrans containing an α(1→2) glucoside bond may make up about30 to 55% of the total oligodextrans.

The synthesis and purification process may be adapted in accordance withthe molar mass of the oligodextrans sought. In particular, the meanmolar mass of the synthesis medium, being higher the higher this ratio.After oligodextrans depends on the sucrose/acceptor ratio in thesynthesis medium, being higher the higher this ratio. After synthesis,the fructose may be kept in the reaction medium or separated by achromatographic ion exchange technique.

Likewise, it should be mentioned that certain additives may be added tothe synthesis medium in order to increase the proportion ofoligodextrans containing an α(1→2) bond, such as certain water-misciblesolvents, such as polyethers, like monoglyme, diglyme, etc., and alsocertain salts such as magnesium chloride, calcium chloride, etc. . . .

Advantageously, if it is desired to eliminate the oligodextrans which donot contain an α(1→2) glucoside bond from the reaction medium, thelatter may be subjected to the action of hydrolases such as theglucoamylase of Aspergillus niger and/or the endodextranase ofPenicillium sp. in order to effect the total hydrolysis of theoligodextrans which do not contain an α(1→2) bond. In contrast, theoligodextrans containing an α(1→2) bond, in particular those with adegree of polymerization of 4, 5, 6 and 7 (abbreviated D.P. 4, D.P.5,D.P.6 and D.P.7), resist hydrolysis. After the action of the hydrolases,the reaction medium contains glucose, fructose and the oligodextranscontaining one or more α(1→2) bonds. The glucose and the fructose may beseparated, if desired, from the oligodextrans by chromatography, forexample with the aid of a cationic exchange resin in the calcium form.The fractions containing the oligodextrans are concentrated underreduced pressure, demineralized, filtered on active charcoal and thendried by lyophilization or by atomization. The final product is a whitepowder which is highly soluble in water (solubility: 70%, m/m) without asugar taste, of neutral pH, and contains 95% by weight or more ofoligodextrans containing one or more α(1→2) glucoside bonds.

The non-limiting examples which follow are given with a view to furtherillustrating the present invention.

EXAMPLE 1 (a) Production and Purification of Glucosyltransferase from L.Mesenteroides B-1299

The L. mesenteroides B-1299 strain is stored in lyophilized form orfrozen in the presence of 10% (v/v) glycerol.

It is cultured in the following culture medium (standard medium):

sucrose: 40 g/l

yeast extract: 20 g/l

dipotassium phosphate: 20 g/l (added in the form of a solution of whichthe pH has been adjusted to 6.9 with the aid of pure orthophosphoricacid)

magnesium sulfate. 7H₂ O: 0.2 g/l

manganese sulfate. H₂ O: 0.01 g/l

calcium chloride. 2H₂ O: 0.02 g/l

sodium chloride: 0.01 g/l

iron sulfate. 7H₂ O: 0.01 g/l

The pH of the culture medium is 6.9. The culture medium and themonopotassium phosphate solution have been previously sterilized by heat(121° C., 20 minutes). If the pH is not adjusted during the culture, themedium acidifies as the growth of the bacterium proceeds. At the end ofthe culture, the pH may reach 4.5. It is preferable to adjust the pH toa higher value of between 5 and 6.5. Adjustment of the pH is carried outwith the aid of 2N sodium hydroxide or of an alkaline solution ofsucrose (400 g/l sucrose; 2N sodium hydroxide). This latter solution isadvantageous because it enables the cellular density of the culture andthe production of the enzyme to be increased slowly.

Additives such as maltose (20 g/l) and the surface-active agent Tween®(1%) promote the excretion of the enzyme in the extracelluar medium in asoluble form.

Table 1 over summarizes the results of three culture tests carried outunder various conditions.

                                      TABLE 1                                     __________________________________________________________________________                                Concentration                                                    Total Soluble                                                                              of insoluble                                                     enzymatic                                                                           extracellular                                                                        matter (cells +                                                                        Insoluble                                CULTURE        activity,                                                                           activity,                                                                            polysaccharides)                                                                       activity,                                CONDITIONS     U/ml  U/ml   mg/ml    U/mg                                     __________________________________________________________________________    Test 1: conical flask                                                                        3.5    0.35  7.9      0.4                                      no adjustment of the                                                          pH of the standard                                                            medium                                                                        Test 2: 2 liter fermenter                                                                     2.75 0.6    4.3      0.5                                      standard medium + maltose                                                     (20 g/l) + Tween ® (1%)                                                   no pH adjustment                                                              Test 3: 2 liter fermenter                                                                    4.0   0.6    8.4      0.4                                      standard medium + maltose                                                     (20 g/l) + Tween ® (1%) +                                                 pH adjustment to 5.6 by                                                       the addition of alkaline                                                      sucrose (400 g/l sucrose:                                                     2N sodium hydroxide)                                                          __________________________________________________________________________

The temperature of the culture medium is 27° C. Stirring is at 500 rpmand aeration is at 1 vvm.

After culturing for 6 h 30, the enzyme concentration in the culturemedium of test 3 is 4 U/ml, of which 0.6 U/ml is in the soluble andextracellular enzymatic form. 85% of the glucosyltransferase isassociated with cells and/or with polysacchardies in the form ofinsoluble aggregates.

After growth, the various forms of the enzyme are extracted from theculture medium by precipitation in the presence of low molecular weightpolyethylene glycol (PEG 1500). All of the enzyme activity precipitatessimultaneously with the polysaccharide produced by the bacterium and thecells, while the PEG 1500 concentration reaches 20% (m/v).

The precipitated fraction is centrifuged (10 minutes, 10,000 g) orrecovered after sedimentation (12 hours, 4° C., in the presence of abacteriostatic agent such as sodium sulfite in a concentration of 1g/liter or sodium benzoate in a concentration of 2 g/liter). After twosuccessive extractions with polyethylene glycol the enzymaticpreparation is lyophilized. The yield from extraction of the enzyme isclose to 100%.

(b) Enzymatic Synthesis of Oligodextrans with Soluble and InsolubleGlucosyltransferase from L. mesenteroides B-1299

The synthesis is carried out under the following conditions:

sucrose: 100 g/l

maltose: 50 g/l

temperature: 30° C.

20 mM sodium acetate buffer, pH 5.2

sodium nitride: 0.5°/oo (bactericide)

enzyme concentration: 0.3 U/ml

After 20 hours reaction, the enzyme is inactivated by heat (30 minutes,80° C.) and the oligodextrans are then analysed by the HPLC methoddescribed above. The results are summarized in Table 2.

                  TABLE 2                                                         ______________________________________                                        Composition of reaction medium after                                          action of soluble and insoluble glucosyltransferase from                      L. mesenteroides B-1299                                                       Sugars                 Concentration, g/l                                     ______________________________________                                        Fructose               52.0                                                   Maltose                15.3                                                   Panose (trisaccharide) 16.6                                                   Oligodextran of D.P. 4 14.9                                                   Oligodextran of D.P. 4 containing                                                                     5.3                                                   an α(1 → 2) bond                                                 Oligodextran of D.P. 5  7.0                                                   Oligodextran of D.P. 5 containing                                                                    18.0                                                   an α(1 → 2) bond                                                 Oligodextran of D.P. >5 containing                                                                    5.9                                                   at least one α(1 → 2) bond                                       Yield of oligodextrans containing                                                                    30%                                                    an α(1 → 2) bond*                                                Yield of the acceptor reaction**                                                                     84%                                                    ______________________________________                                         The yields are expressed in the following way:                                ##STR1##                                                                      ##STR2##                                                                 

(c) Enzymatic Synthesis of Oligodextrans with Soluble and InsolubleGlucosyltransferase from L. Mesenteroides B-1299 in the Presence ofAdditives

Three syntheses 1, 2 and 3 were carried out under the following commonconditions:

sucrose: 100 g/l

maltose: 33 g/l

20 mM sodium acetate buffer, pH 5.2

sodium nitride: 0.5°/oo

enzyme concentration: 1 U/ml

temperature: 30° C. but with the following differences:

synthesis 1: incubation period: 6 hours (without additive)

synthesis 2: incubation period: 23 hours, in the presence of 500 mMmagnesium chloride and 5 mM calcium chloride.

synthesis 3: incubation period: 23 hours, in the presence of 300 mMcalcium chloride.

After the incubation period indicated, the enzyme is inactivated by heat(30 minutes, 80° C.) and the oligodextrans are then analyzed by the HPLCmethod described above. The results are summarized in Table 3.

The rate of the enzymatic reaction is severely slowed in the presence ofhigh concentrations of salts (calcium chloride and magnesium chloride).Nevertheless, a substantial increase in the population of oligodextranscontaining at least one α(1→2) bond is observed relative to the samplesynthesis (carried out without additive).

                  TABLE 3                                                         ______________________________________                                        Composition of the reaction medium after the action of soluble                and insoluble glucosyltransferase from L. mesenteroides B-1299 in             the presence of additives                                                                Synthesis 1                                                                            Synthesis 2                                                                             Synthesis 3                                     ______________________________________                                        Fructose     53.7       47.3      54.6                                        Maltose, leucrose                                                                          7.9        11.0      8.9                                         Panose       6.6        9.7       6.0                                         Oligodextran of                                                                            8.3        7.7       6.0                                         D.P. 4                                                                        Oligodextran of                                                                            1.9        5.9       3.7                                         D.P. 4 containing                                                             an α(1 → 2) bond                                                 Oligodextran of                                                                            5.0        5.4       4.0                                         D.P. 5                                                                        Oligodextran of                                                                            12.2       22.8      17.9                                        D.P. 5 containing                                                             an α(1 → 2) bond                                                 Oligodextran of                                                                            3.8        8.8       11.2                                        D.P. > 5 con-                                                                 taining an                                                                    α(1 → 2) bond                                                    Yield of oligo-                                                                            22%        47%       41%                                         dextrans contain-                                                             ing an α(1 → 2) bond                                             Yield of the 52%        75%       61%                                         acceptor reaction                                                             ______________________________________                                    

EXAMPLE 2 Synthesis of Oligodextrans with the SolubleGlucosyltransferase from L. Mesenteroides B-1299

After centrifuging the culture medium in order to remove the insolublecells and polymers (10,000 g, 20 minutes), the supernatant is subjectedto a liquid-liquid extraction with polyethylene glycol 1,500 inaccordance with the method described in Example 1. The yield from theextraction of the glucosyltransferase is greater than 85%. Thisoperation was carried out twice. The enzyme is then frozen orlyophilized in 20 mM sodium acetate buffer, pH 5.2.

The synthesis is carried out under the following conditions:

sucrose: 100 g/l

maltose: 50 g/l

temperature: 30° C.

20 mM sodium acetate buffer, pH 5.2

enzyme concentration: 0.5 U/ml

reaction time: 4 hours.

After the sucrose has been totally consumed by the glucosyltransferase,the enzyme is denatured by heat (30 minutes, 80° C.). The analysis ofthe oligodextrans present in the reaction medium is carried out by theHPLC method described above. The results are expressed in Table 4.

                  TABLE 4                                                         ______________________________________                                        Composition of the reaction medium after action of the soluble                glucosyltransferase from L. mesenteroides B-1299                              Sugars               Concentration g/l                                        ______________________________________                                        Fructose             50.5                                                     Maltose              11.6                                                     Panose               17.4                                                     Oligodextran of D.P. 4                                                                             19.0                                                     Oligodextran of D.P. 4 containing                                                                   3.1                                                     an α(1 → 2) bond                                                 Oligodextran of D.P. 5                                                                              9.9                                                     Oligodextran of D.P. 5 containing                                                                  18.2                                                     an α(1 → 2) bond                                                 Oligodextrans of D.P. > 5                                                                          13.6                                                     containing at least one α(1 → 2)                                 glucoside bond                                                                Yield of oligodextrans containing                                                                   36%                                                     at least one α(1 → 2) bond                                       Yield of the acceptor reaction                                                                      95%                                                     ______________________________________                                    

The yields do not take account of the oligodextrans of D.P. greater thanor equal to 7, which do not appear on the chromatograms obtained by thismethod.

EXAMPLE 3 Synthesis of oligodextrans with the insolubleglucosyltransferase from L. mesenteroides B-1299

The culture medium is centrifuged (20 minutes, 10,000 m, 4° C.) and theresidue after centrifuging is washed several times with the aid of 20 mMsodium acetate buffer of pH 5.2. The solution is then lyophilized inorder to eliminate all microbial proliferation.

The synthesis of the oligodextrans is carried out under the followingconditions:

sucrose: 100 g/l

maltose: 50 g/l

temperature: 30° C.

20 mM sodium acetate buffer, pH 5.2

sodium nitride: 0.5°/oo

enzyme concentration: 0.9 U/ml

After incubating for 8 hours, the sucrose is totally consumed. Table 5summarizes the composition of the oligodextrans in the synthesis medium.

                  TABLE 5                                                         ______________________________________                                        Composition of the reaction medium after action of the insoluble              glucosyltransferase (associated with cells)                                   from L. mesenteroides B-1299                                                  Sugars                Concentration, g/l                                      ______________________________________                                        Fructose              51.2                                                    Maltose               22.0                                                    Panose                12.8                                                    Oligodextrans of D.P. 4                                                                              9.6                                                    Oligodextrans of D.P. 4 containing                                                                   5.7                                                    an α(1 → 2) bond                                                 Oligodextrans of D.P. 5                                                                              3.7                                                    Oligodextrans of D.P. 5 containing                                                                  13.9                                                    an α(1 → 2) bond                                                 Oligodextrans of D.P. > 5                                                                            3.7                                                    containing at least one α(1 → 2) bond                            Yield of oligodextrans containing                                                                    24%                                                    at least one α(1 → 2) bond                                       Yield of the acceptor reaction                                                                       65%                                                    ______________________________________                                    

EXAMPLE 4

Influence of the concentration of the soluble glucosyltransferase fromL. mesenteroides B-1299 on the synthesis of oligodextrans

Experimental conditions:

sucrose: 100 g/l

maltose: 50 g/l

20 mM sodium acetate buffer, pH 5.2

temperature: 30° C.

Three concentrations of enzyme were used: 0.17, 0.5 and 2 U/ml. Aftertotal consumption of the sucrose, the 3 reaction media were analyzed.The results are identical for the three syntheses, both in respect ofthe yield of the acceptor reaction and in respect of the yield ofoligodextrans containing at least one α(1→2) bond.

EXAMPLE 5

Influence of pH on the enzymatic syntnesis of oligodextrans

Experimental conditions:

sucrose: 100 g/l

maltose: 50 g/l

temperature: 30° C.

enzyme concentration: 1 U/ml

30 mM sodium phosphate citrate buffer

Three pH's were tested: 5.2, 6.0 and 6.4. The activity of the solubleand insoluble enzyme falls slightly when the pH is greater than 6.0.However, no significant difference due to the pH value is observed withregard to to the oligodextran composition of the reaction medium.

EXAMPLE 6

Synthesis of oligodextrans in the presence of a mixture of isomaltoseand isomaltotriose as acceptor with insoluble glucosyltransferase fromL. mesenteroides B-1299

Experimental conditions:

sucrose: 100 g/l

acceptor: 50 g/l composition of the acceptor: isomaltose: 59%isomaltotriose: 36% glucose: 5%

temperature: 30° C.

sodium nitride: 0.5°/oo

20 mM sodium acetate buffer, pH 5.2

enzyme concentration: 1 U/ml

After consumption of the sucrose, the analysis of the reaction medium iscarried out with the aid of the HPLC method described above (Table 6).

                  TABLE 6                                                         ______________________________________                                        Composition of the reaction medium after action of the insoluble              glucosyltransferase from L. mesenteroides B-1299                              Sugars               Concentration, g/l                                       ______________________________________                                        Fructose             53.0                                                     Isomaltose           15.0                                                     Isomaltotriose        6.1                                                     Oligodextrans of D.P. 4 containing                                                                 10.4                                                     an α(1 → 2) bond                                                 Isomaltotetraose      2.4                                                     Oligodextrans of D.P. 5 containing                                                                  9.4                                                     an α(1 → 2) bond                                                 Yield of oligodextrans containing                                                                   20.5%                                                   at least one α(1 → 2) bond                                       Yield of the acceptor reaction                                                                      29.0%                                                   ______________________________________                                    

EXAMPLE 7

Synthesis of oligodextrans in the presence of methyl α-glucoside withthe glucosyltransferase from L. mesenteroides B-1299

Experimental conditions:

sucrose: 100 g/l

methyl α-glucoside: 50 g/l

other conditions: see Example 6.

After consumption of the sucrose, HPLC analysis of the reaction mediumgives the following results (Table 7).

                  TABLE 7                                                         ______________________________________                                        Composition of the reaction medium after action of the insoluble              glucosyltransferase from L. mesenteroides B-1299                              Sugars              Concentration, g/l                                        ______________________________________                                        Fructose            53.0                                                      Methyl α-glucoside                                                                          34.0                                                      Methyl isomaltoside 4.8                                                       Methyl oligodextrans containing                                                                   9.3                                                       an α(1 → 2) bond                                                 Methyl isomaltotrioside                                                                           1.4                                                       Methyl isomaltotetraoside                                                                         0.8                                                       Yield of oligodextrans containing                                                                 10%                                                       at least one α(1 → 2) bond                                       Yield of the acceptor reaction                                                                    26%                                                       ______________________________________                                    

EXAMPLE 8

Enzymatic synthesis of oligodextrans in the presence of variableconcentrations of sucrose with the soluble and insolubleglucosyltransferase from L. mesenteroides B-1299

Experimental conditions:

temperature: 30° C.

20 mM sodium acetate buffer, pH 5.2

sodium nitride: 0.5°/oo

sucrose/maltose ratio: 3

Synthesis 1:

dry material: 20% (w/w)

sucrose: 15% (w/w)

maltose: 5% (w/w)

enzyme concentration: 0.6 U/ml

Synthesis 2:

dry material: 35% (w/w)

sucrose: 26% (w/w)

maltose: 9% (w/w)

enzyme concentration: 1.2 U/ml

Synthesis 3:

dry material: 40% (w/w)

sucrose: 30% (w/w)

maltose: 10% (w/w)

enzyme concentration: 1.8 U/ml

After 21 hours reaction, the enzyme is inactivated by heat (30 minutes,80° C.). The HPLC analysis of the reaction medium is given in Table 8.

                  TABLE 8                                                         ______________________________________                                        Composition of the 3 synthesis media after action of soluble and              insoluble (associated with cells) glucosyltransferase                         from L. mesenteroides B-1299                                                             Concentration, g/l                                                            Synthesis 1                                                                            Synthesis 2                                                                             Synthesis 3                                     ______________________________________                                        Fructose     90.0       150.0     180.0                                       Maltose       8.7        9.7      11.9                                        Leucrose      6.3       13.9      20.4                                        Panose       11.7       17.5      22.9                                        Oligodextran D.P. 4                                                                        14.8       25.5      35.2                                        Oligodextran D.P. 4                                                                         5.7       10.1      13.5                                        containing 1                                                                  α(1 → 2) bond                                                    Oligodextran D.P. 5                                                                        10.6       17.9      20.1                                        Oligodextran D.P. 5                                                                        28.9       44.1      53.4                                        containing 1                                                                  α(1 → 2) bond                                                    Oligodextrans D.P. ≧                                                                15.1       36.6      46.2                                        5 containing at                                                               least 1 α(1 → 2)                                                 bond                                                                          Yield of oligodextrans                                                                      39%        40.5%     38%                                        containing one                                                                α(1 → 2) bond                                                    Yield of the acceptor                                                                       76%        75%       70%                                        reaction                                                                      ______________________________________                                    

The synthesis medium is then subjected to the action of a mixture ofglucoamylase from A. niger (2 U/ml) and endodextranase from Penicilliumsp. (17 U/ml) for 6 hours at 40° C. The enzymatic reaction is stopped byheating at 90° C. for 30 minutes. Glucose and fructose are removed bychromatography on an exchange resin in the calcium form.

The concentration of oligodextrans containing an α(1→2) glucoside bondin the reaction medium is 57 g/l for synthesis 1, 107 g/l for synthesis2 and 122 g/l for synthesis 3.

The distribution of the oligodextrans is as follows:

    ______________________________________                                                  Synthesis 1                                                                            Synthesis 2                                                                              Synthesis 3                                               %        %          %                                               ______________________________________                                        Oligodextrans of                                                                          24         26         30                                          D.P. 4 containing                                                             an α(1 → 2) bond                                                 Oligodextrans of                                                                          69         65         57                                          D.P. 5 containing                                                             an α(1 → 2) bond                                                 Oligodextrans of                                                                           4          5          7                                          D.P. 6 containing                                                             an α(1 → 2) bond                                                 Oligodextrans of                                                                           3          4          6                                          D.P. 7 containing                                                             an α(1 → 2) bond                                                 ______________________________________                                    

EXAMPLE 9

Synthesis of oligodextrans in the presence of a glucose sirup rich inmaltose (Nutriose R 725) by the soluble and insolubleglucosyltransferase from L. mesenteroides B-1299

Nutriose R 725 is a product of Roquette Freres (Lestrem, France), whichhas a high content of maltose and maltotriose.

Means composition of the glucose sirup Nutriose R 725:

dry material: 68% (w/w)

glucose: 1.5%

maltose: 77%

maltotriose: 20%

products with a D.P.≧4: 1.5%

This glucose sirup was used as acceptor under the following conditions:

Synthesis 1:

sucrose concentration: 100 g/l

Nutriose R 725: 68 g/l

sucrose/maltose ratio: 2

enzyme concentration: 0.3 U/ml

other conditions: see Example 8

Synthesis 2:

sucrose concentration: 100 g/l

Nutriose R 725: 42 g/l

sucrose/maltose ratio: 3

enzyme concentration: 0.3 U/ml

other conditions: see Example 8

After incubating for 21 hours, the reaction medium is analyzed by HPLC.The results are summarized in Table 9.

                  TABLE 9                                                         ______________________________________                                        Composition of the reaction medium after action of the soluble                and insoluble glucosyltransferase from L. mesenteroides B-1299                                 Concentration, g/l                                                            Synthesis 1                                                                           Synthesis 2                                          ______________________________________                                        Fructose           53.0      52.0                                             Maltose            18.2      9.7                                              Maltotriose         7.4      4.8                                              Panose             21.7      8.7                                              Oligodextran of D.P. 4                                                                           14.3      8.2                                              Oligodextran of D.P. 4                                                                            4.3      2.0                                              containing an α(1 → 2) bond                                      Oligodextran of D.P. 5                                                                            4.9      4.6                                              Oligodextran of D.P. 5                                                                           15.0      12.9                                             containing an α(1 → 2) bond                                      Oligodextrans of D.P. > 5                                                                         3.8      8.5                                              (containing at least                                                          one α(1 → 2) bond                                                Yield of oligodextrans                                                                            21%      26%                                              containing an α(1 → 2) bond                                      Yield of the acceptor                                                                             74%      60%                                              reaction                                                                      ______________________________________                                    

The synthesis medium is then subjected to the action of a mixture ofglucoamylase from A. niger (2 U/ml) and endodextranase from Penicilliumsp. (17 U/ml) for 6 hours at 40° C. The enzymatic reaction is stopped byheating at 90° C. for 30 minutes. Glucose and fructose are removed bychromatography on an exchange resin in the calcium form.

The concentration of oligodextrans containing an α(1→2) glucoside bondin the reaction medium is 30 g/l for synthesis 1 and 35 g/l forsynthesis 2.

    ______________________________________                                        The distribution of the oligodextrans is as follows:                                           Synthesis 1                                                                           Synthesis 2                                                           %       %                                                    ______________________________________                                        Oligodextrans of D.P. 4                                                                          23        23                                               containing an α(1 → 2) bond                                      Oligodextrans of D.P. 5                                                                          72        70                                               containing an α(1 → 2) bond                                      Oligodextrans of D.P. 6                                                                           3         4                                               containing an α(1 → 2) bond                                      Oligodextrans of D.P. 7                                                                           2         3                                               containing an α(1 → 2) bond                                      ______________________________________                                    

EXAMPLE 10

Hydrolysis of oligodextrans by the glucoamylase from A. niger or amixture of the glucoamylase from A. niger and the endodextranase fromPenicillium sp.

The various reaction media from Examples 1 to 9 contain oligodextrans ofwhich the degree of polymerization varies with the reaction conditions.It is possible to enrich the product in oligodextrans containing anα(1→2) bond of D.P. 4 and 5 by the action of hydrolases which actexclusively on the α(1→6) and α(1→4) bonds.

The glucoamylase from A. niger hydrolyzes the α(1→4) and α(1→6) bonds inoligodextrans starting from the non-reducing end. Its action is blockedby the presence of an α(1→2) glucoside bond whatever its position on theoligodextran. The endodextranase hydrolyzes only α(1→6) glucoside bondsin an endolytic manner on a substrate with a degree of polymerizationgreater than or equal to 3. However, endodextranase does not hydrolyzeoligodextrans of D.P. 4 and D.P. 5 which contain an α(1→2) glucosidebond at the non-reducing end. The conjugated action of these two enzymesenables a product to be obtained which is made up in teh main ofoligodextran of D.P. 4 and oligodextran of D.P. 5.

The fructose liberated in the medium by the action of theglucosyltransferase of L. mesenteroides B-1299 on sucrose and glucoseproduced by the hydrolases can be separated simultaneously bychromatography on an exchange resin in the calcium form, a knowntechnique widely developed industrially for the production of sirupswith a high fructose content.

If a glucoamylase of A. niger is allowed to act on its own, thepopulation of oligodextrans which is obtained has a mean degree ofpolymerization which is higher than in the preceding case; in fact, theaction of the glucoamylase is stopped at the non-reducing end of theoligodextran in the presence of an α(1→2) glucoside bond.

Hydrolysis conditions:

dilution of the synthesis medium to 1/10th

addition of amyloglucosidase NOVO (300 AGU/ml): 3 AGU/ml

addition of dextranase L

AMANO (5000 U/ml): 17 U/ml

temperature: 40° C.

hydrolysis time: 6 hours

After incubating for 6 hours, the two enzymes are inactivated by heat(30 minutes, 100° C.). The HPLC analysis of the two reaction media aftertreatment by the two hydrolases is given in Table 10.

                  TABLE 10                                                        ______________________________________                                                                      Oligodextrans                                             Oligodextrans                                                                          Oligodextrans                                                                            containing an                                             containing an                                                                          containing an                                                                            α(1 → 2)                                     α(1 → 2)                                                                  α(1 → 2)                                                                    bond,                                                     bond, D.P. 4                                                                           bond, D.P. 5                                                                             D.P. > 5                                        ______________________________________                                        Acceptor: maltose                                                                          8 g/l     17 g/l     2 g/l                                       Synthesis conditions:                                                         see Example 3                                                                 Acceptor:   22 g/l      5 g/l     1 g/l                                       isomaltose,                                                                   isomaltotriose                                                                Synthesis conditions:                                                         see Example 6                                                                 ______________________________________                                    

EXAMPLE 11

Synthesis and purification of oligodextrans containing an α(1→2) bondwith the soluble and insoluble glucosyltransferase from Leuconostocmesenteroides B-1299

After having prepared and purified the glucosyltransferase as in Example1(a), the synthesis of the oligodextrans is carried out under thefollowing conditions:

sucrose: 100 g/l

acceptor: Nutriose R 725: 44 g/l (maltose: 33 g/l)

sucrose/maltose ratio: 3

enzyme concentration: 0.3 U/ml

sodium nitride: 0.5%.

pH: 5.6 (adjusted with 1N hydrochloric acid)

temperature: 30° C.

incubation period: 40 hours

reaction volume: 2.5 liters

The enzymatic reaction is stopped by heating at 90° C. for 15 minutes.

After synthesis, the composition of the reaction medium is as follows:

fructose: 49 g/l

leucrose: 6 g/l

maltose: 3 g/l

maltotriose: 7.5 g/l

panose: 12 g/l

oligodextran of D.P. 4 containing an α(1→2) bond: 3 g/l

oligodextran of D.P. 4: 13 g/l

oligodextran of D.P. 5 containing an α(1→2) bond: 15 g/l

oligodextrans of D.P.>5 containing an α(1→2) bond: 10 g/l

The medium also contains oligodextrans of higher D.P. which do notappear in the chromatographic analysis of the synthesis medium.

The synthesis medium is then subjected to the action of a mixture ofglucoamylase from A. niger (3U/ml) and endodextranase from Penicilliumsp. (17 U/ml) for 6 hours at 40° C. The enzymatic reaction is stopped byheating at 90° C. for 30 minutes. Glucose and fructose are removed bychromatography on an exchange resin in the calcium form.

40 g of oligodextrans containing an α(1→2) bond of 94% purity are thusobtained. The distribution of the oligodextrans is as follows:

    ______________________________________                                                             %                                                        ______________________________________                                        glucose, leucrose      6                                                      oligodextrans of D.P. 4 containing                                                                   24                                                     an α(1 → 2) bond                                                 oligodextrans of D.P. 5 containing                                                                   56                                                     an α(1 → 2) bond                                                 oligodextrans of D.P. 6 containing                                                                   7                                                      an α(1 → 2) bond                                                 oligodextrans of D.P. 7 containing                                                                   7                                                      an α(1 → 2) bond                                                 ______________________________________                                    

The preparation is finally lyophilized. After lyophilization the finalproduct is in the form of a white, non-hygroscopic powder without asugar taste and highly soluble in water. Its solubility in water at 20°C. is 70% (m/m).

EXAMPLE 12 Synthesis of α(1→2) oligodextrans in the presence ofoligodextrans of molar mass 1,000 as acceptor

The acceptor used is made up of a mixture of linear oligosaccharidescontaining an α(1→4) bond at the reducing end and α(1→6) bondsexclusively. It is obtained by the action of dextran-saccharase from L.mesenteroides B-512 (F) in the presence of sucrose and maltose.

Experimental conditions Synthesis 1

sucrose: 200 g/l

oligodextrans of molar mass 1,000 (MW: 1,000): 20 g/l

20 mM sodium acetate buffer, pH 5.0

temperature: 23° C.

enzyme concentration: 1 U/ml

Synthesis 2

Synthesis 1 is repeated except that the concentration of the acceptor(oligodextrans of molar mass 1,000) is 40 g/l.

After 40 hours reaction, the enzyme is inactivated by heat treatment ofthe reaction medium (heating at 70° C., 20 minutes). The two reactionmedia are then centrifuged and then subjected to the action of theglucoamylase from Aspergillus niger in order to hydrolyze the α(1→6)glucoside bonds located at the non-reducing end, under the followingconditions:

glucoamylase from A. niger AMG 200 L® (NOVO): 2 AGU/ml

temperature: 40° C.

incubation period: 24 hours

The glucoamylase is then inactivated by heating at 70° C. for 20minutes.

The α(1→2) oligodextrans are then purified by chromatography on an ionexchange resin (Dowex® 50W×4, in the calcium form) in order to removethe mono- and di-saccharides. The α(1→2) oligodextrans are thensubjected to ultrafiltration (cut-off threshold 100,000, AMICON module),demineralized and lyophilized.

In the case of synthesis 1, the mean molar mass by weight (Mw) of theα(1→2) oligodextrans is 1,600. In the case of synthesis 2, the meanmolar mass is 1,400.

We claim:
 1. A process for the preparation of a mixture comprisingoligodextrans a substantial portion of which is comprised ofoligodextrans of the general formula (O-α-D-glucopyranosyl-α(1→2))_(m)(O-α-D-glucopyranosyl-α(1→6))_(n) A, where A is the residue of a sugaracceptor of glucose selected from the group consisting of maltose,isomaltose, isomaltotriose, methyl α-glucoside, and glucose, m is 1 andn is 1, 2 or 3, the position of the α(1→2) glucoside bond being at thenon-reducing end or constituting a branching point of each oligodextran,wherein sucrose and a sugar acceptor of glucose selected from the groupconsisting of maltose, isomaltose, isomaltotriose, methyl α-glucoside,and glucose are brought into contact in the presence ofglucosyltransferase enzyme extracted from the strain B-1299 ofLeuconostoc mesenteroides for approximately 2 to 48 hours in an aqueousmedium.
 2. The process of claim 1, wherein the pH is maintained between4.5 and 6 inclusive during the enzymatic reaction.
 3. The process ofclaim 1, wherein the enzymatic reaction is carried out at a temperatureof about 5° to 45° C.
 4. The process of claim 1, wherein the weightratio of sucrose/sugar acceptor of glucose is between 0.5 and
 10. 5. Theprocess of claim 4, wherein said ratio of sucrose/sugar acceptor ofglucose is between 2 and 4 inclusive.
 6. The process of claim 1, whereinthe enzyme concentration is between 0.2 and 1.0 units/ml of reactionmedium.
 7. The process as claimed in claim 13, which includes thesupplementary step consisting of subjecting the reaction medium, afterremoval or inactivation of the glucosyltransferase enzyme, to the actionof at least one hydrolase enzyme capable of hydrolyzing α-D-(1→6)glycosidic linkages in order to selectively hydrolyse the oligodextranswhich do not contain α(1→2) glucoside bonds present in the reactionmedium.
 8. The process of claim 7, wherein the hydrolase is theglucoamylase from Aspergillus Niger and/or the endodextranase fromPenicillium sp.
 9. The process of claim 1 wherein saidglucosyltransferase enzyme is present in a soluble or insoluble form.